Fig. 1.

Retroviral expression of dominant negative FGFR blocks FGF2 inhibition of OPC differentiation in vitro. A: Replication-incompetent retroviral constructs used to monitor OPC differentiation into mature oligodendrocytes, with or without expression of a dominant negative form of FGFR (FGFRdn). Truncated murine FGFR2 (nucleotides 607–2016) was used to generate FGFRdn, which lacks the cytoplasmic domain required for signaling after ligand binding and homo- or hetero- dimerization with endogenous FGFRs. Each retroviral vector contains 5′ and 3′ long terminal repeat (LTR) regions. The LEGFP control and FGFRdn-FLAG retroviral vectors are generated from a matched plasmid backbone, with either a FLAG epitope tag or enhanced GFP as the reporter. The FGFRdn-FLAG retrovirus generates a fusion protein of FGFRdn and FLAG to facilitate detection of FGFRdn expression and localization. PMXs-FGFRdn was created by inserting the FGFRdn fragment into the PMXs retroviral vector, which contains the cytomegalovirus promoter (CMV) to drive constitutive expression of inserted sequences followed by IRES and then GFP. With the PMXs-FGFRdn retrovirus, the FGFRdn fragment and GFP are generated as a single RNA transcript that is translated as two separate proteins to avoid interference from the GFP reporter. B–D: OPCs infected with each retrovirus expressed the appropriate reporter and differentiation was assessed by immunostaining with O1, an oligodendrocyte marker. Scale bars = 20 μm. Bar for C and D shown in D. B: Immunolabeling of the FLAG epitope tag (red) and O1 antigen (green). Nuclei are labeled with DAPI (blue). Functional block of FGF2 inhibition of OPC differentiation is indicated by acquisition of O1 and elaboration of branched processes. C, D: Immunolabeling of O1 antigen (red) with GFP (green) detection of PMXs (C) or PMXs-FGFRdn (D). In the presence of FGF2, PMXs infected cells remain immature as small, bipolar cells (C) that are not labeled with O1 while cells infected with PMXs-FGFRdn elaborate extensive branched processes and membrane sheets immunolabeled with O1 (D). E: Quantitation of O1 immunolabeling among GFP expressing cells in cultures infected with PMXs or with PMXs-FGFRdn retrovirus. Among cells infected with the PMXs control retrovirus, FGF2 treatment (+) prevents OPC differentiation and acquisition of O1 immunolabeling, as compared with defined medium without FGF2 (−). In contrast, cells infected with PMXs-FGFRdn retrovirus differentiate irregardless of FGF2 treatment, indicating FGFRdn expression blocks endogenous FGFR responsiveness to FGF2. Values shown are the mean of three independent experiments, with at least 300 cells counted for each retrovirus condition, and error bars denote standard error of the proportion. The asterisk (*) indicates a significance difference of P < 0.001 for PMXs versus PMXs-FGFRdn in the presence of FGF2.